Posttranslational modifications that regulate the trafficking, availability, or activity of secreted ligands are emerging as critical control points in many signaling pathways. Prior to secretion, most Wnt ligands undergo glycosylation and lipid modification in the endoplasmic reticulum before they are transported to the Golgi complex. The transmembrane protein Wntless (Wls) facilitates the transport of Wnts from the trans-Golgi network to the plasma membrane, from which the Wnts are secreted and Wls is recycled back to the Golgi. Six of the seven Wnt proteins in the fruit fly Drosophila melanogaster are lipidated and depend on Wls for secretion, whereas WntD neither is lipidated nor requires Wls for secretion. Herr and Basler investigated the link between lipid modification and Wls-dependent secretion for each of the Drosophila Wnts. Expression of any of the Wls-dependent Wnts caused Wls to relocate to the Golgi in wild-type (WT) wing disc cells; these Wnts accumulated intracellularly in wls mutant wing disc cells, and each was glycosylated in cultured cells. In contrast, WntD did not induce Wls relocation, was secreted from wls mutant cells, and was not glycosylated. Mutant versions of Wg (one of the Wls-dependent Wnts) that lacked glycosylation sites mimicked WT Wg, which implied that glycosylation did not affect Wls-mediated secretion. O-linked acylation of Wg at Ser239 by the acyltransferase Porcupine (Por) is required for subsequent S-palmitoylation of Cys93 and for signaling activity. Both WT Wg and a version of Wg that lacked the S-palmitoylation site (WgC93A) accumulated in wls mutant wing disc cells, but a version of Wg that lacked the acylation site (WgS239A) was secreted, so lipid modification of Ser239 correlated with dependence on Wls for secretion. Each of the other Wls-dependent Wnts retain an equivalent conserved Ser that is not present in WntD. Expression of any of the Wls-dependent Wnts failed to induce Wls relocalization to the Golgi in por mutant clones, and expression of mutant versions of these Wnts lacking the acylation site also failed to induce Golgi relocalization in WT wing disc cells. The authors conclude that lipid modification of Wnts renders their secretion dependent on Wls and that Wnts that are not lipidated do not depend on Wls for secretion.